Analysis of the Phlebiopsis Gigantea Genome, Transcriptome and Secretome Provides Insight Into Its Pioneer Colonization Strategies of Wood

Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

Chiaki Hori1, Takuya Ishida1, Kiyohiko Igarashi1, Masahiro Samejima1, Hitoshi Suzuki2, Emma Master2, Patricia Ferreira3, Francisco J. Ruiz-Duen˜ as4, Benjamin Held5, Paulo Canessa6, Luis F. Larrondo6, Monika Schmoll7, Irina S. Druzhinina8, Christian P. Kubicek8, Jill A. Gaskell9, Phil Kersten9, Franz St. John9, Jeremy Glasner10, Grzegorz Sabat10, Sandra Splinter BonDurant10, Khajamohiddin Syed11, Jagjit Yadav11, Anthony C. Mgbeahuruike12, Andriy Kovalchuk12, Fred O. Asiegbu12, Gerald Lackner13, Dirk Hoffmeister13, Jorge Rencoret14, Ana Gutie´rrez14, Hui Sun15, Erika Lindquist15, Kerrie Barry15, Robert Riley15, Igor V. Grigoriev15, Bernard Henrissat16, Ursula Ku¨ es17, Randy M. Berka18, Angel T. Martıńez4, Sarah F. Covert19, Robert A. Blanchette5, Daniel Cullen9* 1 Department of Biomaterials Sciences, University of Tokyo, Tokyo, Japan, 2 Department of Chemical Engineering, University of Toronto, Toronto, Ontario, Canada, 3 Department of Biochemistry and Molecular and Cellular Biology and Institute of Biocomputation and Physics of Complex Systems, University of Zaragoza, Zaragoza, Spain, 4 Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain, 5 Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota, United States of America, 6 Millennium Nucleus for Fungal Integrative and Synthetic Biology and Departamento de Gene´tica Molecular y Microbiologıá, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile, 7 Health and Environment Department, Austrian Institute of Technology GmbH, Tulin, Austria, 8 Austrian Center of Industrial Biotechnology and Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria, 9 USDA, Forest Products Laboratory, Madison, Wisconsin, United States of America, 10 University of Wisconsin Biotechnology Center, Madison, Wisconsin, United States of America, 11 Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio, United States of America, 12 Department of Forest Sciences, University of Helsinki, Helsinki, Finland, 13 Department of Pharmaceutical Biology at the Hans-Kno¨ll-Institute, Friedrich-Schiller-University, Jena, Germany, 14 Instituto de Recursos Naturales y Agrobiologia de Sevilla, CSIC, Seville, Spain, 15 US Department of Energy Joint Genome Institute, Walnut Creek, California, United States of America, 16 Architecture et Fonction des Macromolećules Biologiques, Unite´ Mixte de Recherche 7257, Aix-Marseille Universite´, Centre National de la Recherche Scientifique, Marseille, France, 17 Molecular Wood Biotechnology and Technical Mycology, Bu¨sgen-Institute, Georg-August University Go¨ttingen, Go¨ttingen, Germany, 18 Novozymes, Inc., Davis, California, United States of America, 19 Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia, United States of America

Abstract Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh- cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea’s extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

Citation: Hori C, Ishida T, Igarashi K, Samejima M, Suzuki H, et al. (2014) Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood. PLoS Genet 10(12): e1004759. doi:10.1371/journal.pgen.1004759 Editor: Gregory P. Copenhaver, The University of North Carolina at Chapel Hill, United States of America Received April 15, 2014; Accepted September 16, 2014; Published December 4, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The major portions of this work were performed under US Department of Agriculture Cooperative State, Research, Education, and Extension Service Grant 2007-35504-18257 (to DC and RAB). The US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231. This work was also supported by the HIPOP (BIO2011-26694) project of the Spanish Ministry of Economy and Competitiveness (MINECO) (to FJRD), the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013-.3.3-04-613549) European projects (to ATM), and the Chilean National Fund for Scientific and Technological Development Grant 1131030 (to LFL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]

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Author Summary In addition to peroxidases, laccases have been implicated in lignin degradation [24–26]. To date, multiple laccase isozymes The wood decay fungus Phlebiopsis gigantea degrades all and/or the corresponding genes have been characterized from components of plant cell walls and is uniquely able to most white-rot fungi except P. chrysosporium, an efficient rapidly colonize freshly exposed conifer sapwood. Howev- lignocellulose degrader that lacks such enzymes [27–29]. The er, mechanisms underlying its conversion of lignocellulose mechanism(s) by which laccases might degrade lignin remain and resinous extractives have not been explored. We unclear as the enzyme lacks sufficient oxidation potential to cleave report here analyses of the genetic repertoire, transcrip- non-phenolic linkages within the polymer. Interestingly, laccase tome and secretome of P. gigantea. Numerous highly activity has not been reported in P. gigantea. expressed hydrolases, together with lytic polysaccharide Additional ‘auxiliary activities’ [30] commonly ascribed to monooxygenases were implicated in P. gigantea’s attack ligninolytic systems include extracellular enzymes capable of on cellulose, and an array of ligninolytic peroxidases and generating H O . These enzymes may be physiologically coupled auxiliary enzymes were also identified. Comparisons of 2 2 woody substrates with and without extractives revealed to peroxidases. Among them, aryl-alcohol oxidase (AAO), differentially expressed genes predicted to be involved in methanol oxidase (MOX), pyranose 2-oxidase (P2O), and copper the transformation of resin. These expression patterns are radical oxidases (such as glyoxal oxidase, GLX) have been likely key to the pioneer colonization of conifers by P. extensively studied. With the exception of P2O [31], none of gigantea. these activities have been reported in P. gigantea cultures. In short, the repertoire of extracellular enzymes produced by P. Introduction gigantea is largely unknown, and its mechanism(s) for cell wall degradation remain unexplored. The most abundant source of terrestrial carbon is plant biomass, Beyond extracellular systems, the complete degradation of composed primarily of cellulose, hemicellulose, and lignin. lignin requires many intracellular enzymes for the complete Numerous microbes utilize cellulose and hemicellulose, but a mineralization of monomers to CO2 and H2O. Examples of much smaller group of filamentous fungi has the capacity to enzymes that have been characterized from P. chrysosporium degrade lignin, the most recalcitrant component of plant cell walls. include cytochromes P450 (CYPs) [32–34], glutathione transfer- Uniquely, such ‘white-rot’ fungi efficiently depolymerize lignin to ases [35], and aryl alcohol dehydrogenase (AAD) [36]. The role of access cell wall carbohydrates for carbon and energy sources. As such enzymes in P. gigantea, if any, is unknown. such, white-rot fungi play a key role in the carbon cycle. Herein, we report analysis of the P. gigantea draft genome. White-rot basidiomycetes may differ in their substrate prefer- Gene annotation, transcriptome analyses and secretome profiles ence and morphological patterns of decay (for review see [1,2]). identified numerous genes involved in lignocellulose degradation The majority of lignin-degrading fungi, including Phanerochaete and in the metabolism of conifer extractives. chrysosporium and Ceriporiopsis subvermispora, are unable to colonize freshly cut wood unless inhibitory compounds (extrac- Results tives) are removed or transformed [2–5]. A few basidiomycetes, including Phlebiopsis gigantea, are pioneer colonizers of softwood Genome assembly and annotation because they tolerate and utilize resinous extractives (e.g., resin Following an assessment of wood decay properties (Figure 2), P. acids, triglycerides, long chain fatty acids, see Figure 1) which gigantea single basidiospore strain 5–6 was selected for sequencing cause pitch deposits in paper pulp manufacturing [6]. It is this using Illumina reads assembled with AllPathsLG. Genome size unusual capability that also led to the development of P. gigantea was estimated to be approximately 30 Mbp (Text S1), somewhat as a biocontrol agent against subsequent colonization of cut lower than closely related members of the ‘Phlebia clade’ [23,37] stumps by the root rot pathogen Heterobasidium annosum sensu such as C. subvermispora (39 Mbp) and P. chrysosporium lato (now considered several species) [7,8] and of harvested wood (35 Mbp) [22,27]. Aided by 17,915 mapped EST clusters, the by blue stain fungi [9,10]. It seems likely that when applied to JGI annotation pipeline predicted 11,891 genes. Proteins were freshly cut wood, P. gigantea is able to rapidly metabolize assigned to 6412, 5615, 6932 and 2253 KOG categories, GO accessible extractives and hemicellulose. As the hyphae continue to terms, pfam domains and EC numbers, respectively. Significant invade tracheids and ray parenchyma cells, the more recalcitrant synteny with P. chrysosporium was observed (Figure S1). Detailed cell wall polymers (cellulose, lignin; Figure 1) are eroded. Little is information on the assembly and annotations is available via the known of how some white-rot fungi degrade conifer extractives JGI portal MycoCosm [38]. [11,12] or interact with other fungi such as H. annosum [13]. White-rot fungi degrade major cell wall polymers through Gene families concerted action of hydrolytic and oxidative enzymes (reviewed in Principal component analysis (PCA), based on 73 and 12 [14,15]). Cellulose is attacked by a combination of exo- families of carbohydrate active enzymes (CAZys, [16]) and cellobiohydrolases and endoglucanases assigned to glycoside auxiliary activities (AAs), [30]), respectively, clustered P. gigantea hydrolase families GH5, GH6, GH7 and possibly GH9, GH12, with other efficient lignin degraders ([39], Figures 3A and S2). GH44 and GH45 [16,17]. In addition to these hydrolases, recent Gene numbers were extracted from 21 fungal genomes and evidence strongly supports the involvement of lytic polysaccharide excluded genes encoding putative GMC oxidases such as monooxygenases (LPMOs) in cellulose degradation [18–20]. methanol oxidase, alcohol oxidase and glucose oxidase (Dataset Lignin degradation is catalyzed by an array of oxidative enzymes, S1). Highest contribution of PC1 (50% of variance separating especially lignin peroxidase (LiP), manganese peroxidase (MnP) white-rot and brown-rot fungi) and PC2 (13.0% of variance)) and versatile peroxidase (VP) belonging to class II of the plant- values were those genes associated with degradation of plant cell fungal-prokaryotic peroxidase superfamily. Recent genome inves- wall polysaccharides and lignin, respectively (Text S1). Hierarchi- tigations reveal that all efficient lignin degraders possess some cal clustering analysis with this dataset also categorized P. gigantea combination of these class II ligninolytic peroxidases [21,22]. In P. into a clade of white-rot fungi that included the polypore P. gigantea, four MnP sequences were previously identified [23]. chrysosporium. The precise number and distribution of P. gigantea

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Figure 1. Schematic representations of lignocellulose components in cell walls of pine wood. Panel A: The extractives (long chain fatty acids, triglycerides, resin acids and terpenes) are found primarily in the resin ducts, but damage to pine wood causes the release of these compounds across wounded areas. Panel B: In tracheid cell walls, the amorphous, phenylpropanoid polymer lignin (brown) form a matrix around the more structured carbohydrate polymers, hemicellulose (yellow and green) and cellulose (blue). doi:10.1371/journal.pgen.1004759.g001 genes likely involved in lignocellulose degradation were similar, family includes 1,2-a-fucosidases that hydrolyze the a-Fuc-1,2-Gal but not identical, to other polypores such as P. chrysosporium and linkages in plant xyloglucans. C. subvermispora (Figure 4). Like P. chrysosporium and Phaner- The P. gigantea genome includes representatives for all the ochaete flavido-alba, P. gigantea had no laccase sensu stricto peroxidase families reported in basidiomycetes, including LiP, genes. Interestingly, while both P. gigantea and the white-rot MnP, heme-thiolate peroxidases, and dye-decolorizing type Russulales H. annosum are adapted to colonization of conifers, peroxidases (DyP), with the only exception of VP (Text S1; significant numbers of laccase sensu stricto genes were only Figures S8–S13). MnP gene expansion is similar to that found in observed in H. annosum (Figure 4). This important conifer the C. subvermispora and H. annosum genomes. Beyond class II pathogen also lacked GLX, LiP and representatives of GH5 peroxidases and multicopper oxidases (MCOs), genes encoding subfamiles 15 and 31. auxiliary enzymes involved in ligninolysis were also found such as With regard to hemicellulose degradation, the genomes of GMC oxidoreductases (Figures S14–S19; Table S5) and copper conifer-adapted P. gigantea and H. annosum revealed increased radical oxidases (CRO, Figure 4; Table S4). Among the latter numbers of genes involved in pectin degradation such as GH28 group, GLX is coupled to P. chrysosporium LiPs via extracellular polygalacturonase, CE8 pectin methylesterase and CE12 rham- H2O2 generation [41]. Consistent with this physiological connec- nogalacturonan acetylesterase (Figure 4). The major hemicellulose tion, the P. gigantea genome features both GLX- and LiP- of conifer is galactoglucomannan ([40], Figure 1) but, in the case encoding genes. GMC genes encoding putative AAO, MOX and of mannan degradation, no significant increase in genes encoding glucose oxidase (GOX) may also be involved in H2O2 production GH2 b-mannosidase, GH5_7 endo-mannanase and GH27 a- by oxidation of low molecular weight aliphatic and aromatic galactosidase was observed relative to other wood decay fungi alcohols. The P2O gene (protein model Phlgi1_130349) lies (Figure 4). Similarly, no significant differences in the number of immediately adjacent to a putative pyranosone dehydratase genes involved in arabinoglucuronoxylan hydrolysis were identi- (Phlgi1_16096) gene. This arrangement is conserved in several fied, except for two transcriptionally convergent GH11 genes wood decay fungi and, in addition to peroxide generation, suggests present in P. gigantea (Text S1). Encoding putative endo-1,4-b- a route for conversion of glucose to the pyrone antibiotic, xylanases, wood decay fungi typically harbor one or no GH11 cortalcerone [42,43]. Genes encoding AAD, members of the genes. Auricularia delicata is another exception with three of these zinc-type alcohol dehydrogenase superfamily [44], are also endoxylanases. Also unusual among white-rot fungi, none of the P. abundant in P. gigantea. Relatively few genes were predicted to gigantea protein models were assigned to GH95 (Dataset S1). This encode CYPs which are generally considered important in the

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Figure 2. Wood decay characteristics. Comparative weight loss of parental strain 11061 and single basidiospore derivatives on colonized loblolly pine wood (Pinus taeda) wood wafers were determined after 4, 8 and 12 weeks incubation (bottom left panel) as described in Methods. Single basidiospore strain 5–6 also aggressively decayed birch and spruce (Text S1) and was selected for sequencing. Upper panels show scanning electron microscopy [68] of radial (left) and transverse (right) sections of pine wood tracheids that were substantially eroded or completely degraded by P. gigantea strain 5–6 by week twelve. Transverse section of sound wood (bottom photo) provides comparison. (Bar = 40 mm). doi:10.1371/journal.pgen.1004759.g002 intracellular metabolism of lignin derivatives and related aromatic and CYP505 clans of cytochrome P450s are associated with compounds (Figure S19; Dataset S2). degradation of fatty acids and alkanes. Relative to other white-rot The repertoire of P. gigantea genes contrasts sharply with that fungi, P. gigantea had a slightly greater number of CYP52- of brown-rot polypores, such as Postia placenta [45], which lack encoding genes whereas CYP505 gene numbers were similar ligninolytic class II peroxidases, cellobiohydrolases (GH6, GH7), (Figure 4; Dataset S1; Figures S31, S32; Tables S13–S15). and endoglucanases fused to cellulose binding modules [21,46] P. gigantea also diverges from other Agaricomycetes with (Figure 4). Unlike P. gigantea and other white-rot fungi, brown- respect to genes encoding proteins that are more distantly rot fungi often lack genes encoding cellobiose dehydrogenase connected to lignocellulose degradation, including hydrophobins (CDH) and have relatively few lytic polysaccharide monooxygen- (Figures S33 and S34; Tables S17–S19), transporters (Table S20) ase genes (LPMOs). Formerly classified as GH61 ‘hydrolases’, the and non laccase MCOs (Figure S20). Detailed analyses are LPMOs are now known to be copper-dependent monooxygenases provided for CAZys (Tables S7–S10; Figures S22–S30; Dataset [18–20] capable of enhancing cellulose attack by CDH and S1), peroxidases (Figures S8–S13), auxiliary proteins, cytochrome cellobiohydrolase (CBH) [47,48]. With the exception of Gloeo- P450s (Figures S31–S32; Table S13–S15), potential regulatory phyllum trabeum, genes encoding GH74 enzymes have not been genes (Figures S4–S7; Tables S3, S11–S12) and genes involved in found in brown-rot fungi. Two such xyloglucanase genes were secondary metabolite synthesis (Table S16). identified in P. gigantea (Text S1). In contrast to analysis of genes involved in lignocellulose Differential gene expression of P. gigantea in response to degradation (Figure 3A), white-rot and brown-rot fungi were not substrate clearly separated by principal component analysis of 14 enzymes Transcript levels were determined in cultures in which the sole involved in lipid metabolism (Figures 3B and S3). However, P. carbon source was glucose (Glc), freshly harvested loblolly pine gigantea was grouped near B. adusta and P. carnosa. These wood (Pinus taeda; LP) extracted with acetone (ELP), or freshly associations seem in line with the preferential colonization of harvested but not extracted loblolly pine wood (NELP) (Text S1). softwood substrates by P. carnosa [49] and with the efficient GC-MS analysis [51] identified the major extract components as degradation of conifer extractives by B. adusta culture superna- resin acids (46%), triglycerides (13%) and fatty acids (11%) (Text tants [50].The highest contribution to PC1 (26.0% variance) and S1; Figure S35; Table S21). PC2 (6.8% variance) were aldehyde dehydrogenase and long chain Excluding genes with relatively low transcript levels (RPKM fatty acid CoA ligase, respectively (Figures 3A and S3, Text S1). values ,10) in LP-containing media, transcripts of 187 genes were Also potentially involved in intracellular lipid metabolism, CYP52 increased.2-fold (p,0.05) in NELP or ELP relative to Glc. Of

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Figure 3. Comparative analysis of gene repertoires associated with degradation of plant cell wall polymers and extractives in 21 fungal genomes. (A) Principal component analysis (PCA) of 21 fungi using 73 CAZy and 12 AA families (Dataset S1). GMC oxidoreductases methanol oxidase, glucose oxidase and aryl alcohol oxidase were excluded because confident functional assignments could not be made and/or their inclusion did not contribute to separation of white- and brown-rot species. (B) PCA of 21 fungi using genes encoding 14 enzymes involved in lipid metabolism (KEGG reference pathway 00071, Dataset S1). There is no significant segregation of white-rot and brown-rot fungi although P. gigantea was positioned in the third quadrant with B. adusta and P. carnosa. Symbols for white rot and brown rot fungi appear in blue and red, respectively. Tremella mesenterica is a mycoparasite. For raw data and contributions of the top 20 families see Dataset S1, Text S1 and Figures S2 and S3. doi:10.1371/journal.pgen.1004759.g003 those Glc-derived transcripts with RPKM values.10, 146 genes walls and/or secreted via ‘non-classical’ mechanisms ([54]; www. had higher transcripts in Glc relative to NELP or ELP (Figure 5; cbs.dtu.dk/services/SecretomeP). Still other peptides correspond Dataset S2). to true intracellular proteins released by cell lysis, e.g. ribosomal Mass spectrometry (nanoLC-MS/MS) identified extracellular proteins (Dataset S2). peptides corresponding to a total of 319 gene products in NELP Glycoside hydrolase gene expression was heavily influenced by and ELP cultures (Dataset S2). Most proteins were observed in media composition with transcripts corresponding to 76 genes both NELP and ELP culture filtrates, which contained 294 and increasing.2-fold in NELP- or ELP-containing media relative to 268 proteins, respectively. Approximate protein abundance, glucose medium (Figure 6). Some of these genes were highly expressed as the exponentially modified protein abundance index expressed with RPKM values well over 100. For example, (emPAI) [52], varied substantially within samples. As expected, transcript and peptide levels matching GH7 cellobiohydrolase gene products with predicted secretion signals and high transcript (CBH1; model Phlgi1_34136) were among the ten most highly levels were often detected. Other detected proteins (e.g. MOX expressed genes (Table 1). Indicative of a complete cellulolytic model Phlgi1_120749; [53]) may be loosely associated with cell system, this CBH1 was accompanied by upregulated transcripts

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Figure 4. Number of genes identified in white rot fungi P. gigantea (Phlgi), P. chrysosporium (Phach)[27], C. subvermispora (Cersu)[22], and H. annosum (Hetan)[75], and the brown rot fungus P. placenta (Pospl)[45]. CROs were distinguished as previously described [76]. Lytic polysaccharide monooxygenases were formerly classified as GH61 within the CAZy system (http://www.cazy.org/; [16]). Glycoside hydrolase family GH5 was subdivided as described [77] (Figure S22). doi:10.1371/journal.pgen.1004759.g004 and extracellular proteins corresponding to another CBH1 did not observe high expression of class II peroxidases under the (Phlgi1_13298), a GH6 family member CBH2 (Phlgi1_17701) conditions tested (Dataset S2). On the other hand, a DyP and GH5_5 b-1,4 endoglucanases (EGs; Phlgi1_86144, (Phlgi1_85295) was significantly upregulated in certain LP- Phlgi1_84111), all of which feature a family 1 carbohydrate containing media (Table 1). The importance of these peroxidases binding module (CBM1). Also highly expressed were putative b- is further supported by the high protein levels of another DyP, glucosidases (Phlgi1_127564, Phlgi1_18210) and a GH12 Phlgi1_122124. Specifically, the latter protein showed emPAI (Phlgi1_34479). Other glycoside hydrolases likely involved in values.17 after 5 days growth on LP media and, relative to Glc degradation of cell wall hemicelluloses include GH5_7 endoman- medium, its transcript ratios were.5-fold higher (p,0.04) nanases (Phlgi1_97727, Phlgi1_110296), a GH74 xyloglucanase (Dataset S2). High DyP gene expression has been observed in (Phlgi1_98770), a GH27 a-galactosidase (Phlgi1_72848) and a white-rot fungi Trametes versicolor and Dichomitus squalens [21], GH10 endoxylanase (Phlgi1_85016). but no genes for these proteins are present in P. chrysosporium Expression of oxidative enzymes implicated in lignocellulose and C. subvermispora (Figure 4). The P. gigantea DyP degradation was also influenced by growth on LP-media (NELP or (Phlgi1_122124) was also abundant in media containing micro- ELP) relative to Glc-containing media. Transcripts corresponding crystalline cellulose (Avicel) as the sole carbon source (Dataset S2). to five LPMO-encoding genes showed significant regulation (P, To identify enzymes involved in tolerance to and/or degrada- 0.01) in LP-medium, and three LPMO proteins were detected tion of extractives, comparisons were made of gene expression in (Phlgi1_227588, Phlgi1_227560, Phlgi1_37310). An AAD-like ground loblolly pine wood that had been extensively extracted oxidoreductase (Phlgi1_30343), possibly involved in the transfor- with acetone (ELP) versus non-extracted loblolly pine wood mation of lignin metabolites, was also upregulated. However, we (NELP) (Figure 7A). In general, this treatment had little impact on

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Figure 5. P. gigantea transcriptome. Scatterplots show the distribution of RNA-seq RPKM values (log2) for 11,376 P. gigantea genes when grown on basal salts containing A, acetone-extracted loblolly pine wood (ELP) or B, non-extracted loblolly pine wood (NELP) relative to glucose (Glc). Plot lines define 2-fold borders and best fit regression. Darkened points represent 164 (A) and 145 (B) transcripts accumulating.4-fold at p,0.01. Venn diagram (C) illustrates genes with RPKM signals.10 and upregulated.4-fold in NELP or ELP relative to Glc. doi:10.1371/journal.pgen.1004759.g005 gene expression. For example, glycoside hydrolase transcript and factors). Integration of transcript profiles of genes involved in protein patterns showed only minor differences (Figure 8). intracellular lipid and oxalate metabolism, together with TCA and Nevertheless, transcripts corresponding to 22 genes showed glyoxylate cycles, strongly supports a central role for b-oxidation in significantly increased levels (.4-fold; p,0.01) in NELP relative triglyceride and terpenoid transformation by P. gigantea (Fig- to ELP (Figure 7B; Table 2). Of particular interest were genes ure 9). potentially involved in metabolism of resin acids (e.g. CYPs; [55]), Relaxing the transcript fold-change threshold (.2-fold; p,0.01) in altering the accessibility of cell wall components (e.g., an and focusing on mass spectrometry-identified proteins revealed 14 endoxylanase), and in regulating gene expression (e.g. proteins additional genes potentially involved in metabolism and/or containing putative Zn finger domains or HMG-Box transcription tolerance to loblolly pine wood extractives (Table 3).Among these

Figure 6. Number and expression of genes likely involved in lignocellulose degradation. The number of genes encoding mass spectrometry-identified proteins was limited to those matching$2 unique peptides after 5–9 days growth in media containing NELP or ELP. RPKM values.100 for RNA derived from these cultures were arbitrarily selected as the threshold for high transcript levels. Genes designated as ‘regulated’ showed significant accumulation (p,0.05;.2-fold) in NELP or ELP relative to glucose containing media. Methods and complete data are presented in Text S1 and Dataset S2. doi:10.1371/journal.pgen.1004759.g006

emPAI value of filtrates from pine cultures RNA-seq 5 Day Transcript ratios (R) & probabilities (P)

NELP ELP RPKM value NELP/Glc NELP/ELP ELP/Glc

PRO id Putative function 5 Day 7 Day 9 Day 5 Day 7 Day 9 Day NELP ELP Glc Prob R Prob R Prob R

Fifteen highly expressed (.100 RPKM) genes exhibiting significant transcript accumulation (p,0.01;.4-fold) in NELP medium relative to Glc medium: 79150 GH61 LPMO 114 51 2 ,0.01 58.6 0.286 2.2 0.021 26.2 74623 Carotenoid ester lipase 0.00 0.39 0.18 0.36 0.00 0.00 313 159 8 ,0.01 40.5 0.046 2.0 0.013 20.7 128120 GH15 Glucoamylase 3.06 1.45 0.53 2.35 4.76 4.75 170 47 5 ,0.01 32.2 0.010 3.6 0.023 8.9 20514 Cytochrome P450* 368 30 14 ,0.01 27.2 ,0.01 12.3 0.037 2.2 88425 GH55 b- 1,3 glucanase 0.76 1.48 0.68 0.55 0.81 0.74 149 49 6 ,0.01 26.3 0.120 3.0 0.057 8.7 109878 SDH 510 1000 20 ,0.01 26.0 0.474 0.5 0.025 51.0 88507 GH18 Chitinase* 409 88 19 ,0.01 21.6 ,0.01 4.7 0.046 4.6 123837 Zn finger domain* 8223 1745 436 ,0.01 18.9 ,0.01 4.7 0.057 4.0 36218 Hypothetical 2.48 0.57 0.19 0.85 0.00 0.00 3992 2334 227 ,0.01 17.6 0.209 1.7 0.014 10.3 123273 Epoxide hydrolase 139 112 10 ,0.01 13.8 0.767 1.2 0.044 11.2 125213 GH61 (LPMO)* 1291 306 109 ,0.01 11.8 ,0.01 4.2 0.058 2.8 30343 AAD-like OR1 308 168 34 ,0.01 9.2 0.364 1.8 0.083 5.0 36293 Hypothetical 371 352 46 ,0.01 8.1 0.885 1.1 0.012 7.7 105051 Peptidase M 176 51 24 ,0.01 7.3 ,0.01 3.4 0.065 2.1 107268 Hypothetical 110 69 15 ,0.01 7.3 0.035 1.6 0.013 4.6 Thirty highly expressed genes (RPKM.100) exhibiting significant transcript accumulation (p,0.01;.4-fold) in NELP and ELP medium relative to Glc medium: 34136 GH7 Cellobiohydrolase 16.85 5.77 2.91 35.76 0.82 2.91 3927 2931 40 ,0.01 97.7 0.218 1.3 ,0.01 72.9 98430 Hexose transporter 0.08 0.06 0.06 0.37 0.00 0.00 2213 1794 63 ,0.01 35.4 0.323 1.2 ,0.01 28.7 19028 Lipase* 6.86 1.20 0.76 1.79 1.92 1.34 1903 274 22 ,0.01 87.7 ,0.01 7.0 ,0.01 12.6

110296 GH5-7 Mannanase 5.29 24.92 19.23 2.12 0.68 0.87 1448 1243 15 ,0.01 97.0 0.597 1.2 ,0.01 83.3 gigantea Phlebiopsis 101670 Peptidase M35 1.39 0.54 0.28 0.80 0.00 0.00 1232 583 22 ,0.01 56.8 0.146 2.1 ,0.01 26.9 69030 Aquaporin 1080 518 18 ,0.01 58.7 0.011 2.1 ,0.01 28.2 17701 GH6 Cellobiohydrolase 3.68 2.17 1.06 2.92 0.21 0.19 699 436 9 ,0.01 79.9 0.244 1.6 ,0.01 49.8 99876 CDH 5.31 1.37 0.63 5.63 0.12 0.23 602 327 12 ,0.01 50.4 0.065 1.8 ,0.01 27.4 97727 GH5-7 Mannanase 1.16 0.60 0.24 0.89 0.00 0.00 538 721 16 ,0.01 34.5 0.130 0.7 ,0.01 46.2 ooiaino Conifers of Colonization 13298 GH7 Cellobiohydrolase 1.00 1.05 0.59 0.64 0.00 0.17 418 296 9 ,0.01 45.3 0.215 1.4 ,0.01 32.1 98770 GH74 Xyloglucanase 3.53 1.82 0.76 4.36 0.23 0.69 373 446 9 ,0.01 43.4 0.307 0.8 ,0.01 51.9 227588 GH61 (LPMO)2 0.61 0.44 0.09 0.19 0.00 0.00 367 592 11 ,0.01 34.5 0.276 0.6 ,0.01 55.7 18264 GH61 (LPMO) 344 150 4 ,0.01 92.2 0.209 2.3 ,0.01 40.2 127564 GH3 b-glucosidase 5.02 5.89 4.87 4.84 0.48 0.33 279 191 8 ,0.01 35.6 0.134 1.5 ,0.01 24.4 72848 GH27 a-galactosidase 0.70 1.25 0.53 0.74 0.00 0.00 244 232 28 ,0.01 8.8 0.850 1.0 ,0.01 8.4 LSGntc w.lseeisog9Dcme 04|Vlm 0|Ise1 e1004759 | 12 Issue | 10 Volume | 2014 December 9 www.plosgenetics.org | Genetics PLOS Table 1. Cont.

emPAI value of filtrates from pine cultures RNA-seq 5 Day Transcript ratios (R) & probabilities (P)

NELP ELP RPKM value NELP/Glc NELP/ELP ELP/Glc

PRO id Putative function 5 Day 7 Day 9 Day 5 Day 7 Day 9 Day NELP ELP Glc Prob R Prob R Prob R

86144 GH5-5 Endoglucanase 5.89 1.29 0.61 2.69 0.00 0.00 238 223 4 ,0.01 62.2 0.901 1.1 ,0.01 58.4 23523 Hypothetical 216 285 50 ,0.01 4.3 0.169 0.8 ,0.01 5.7 34479 GH12 Endoglucanase 197 114 2 ,0.01 87.0 0.210 1.7 ,0.01 50.4 85016 GH10 Endoxylanase 34.64 19.63 4.82 18.22 0.75 2.62 175 173 3 ,0.01 68.0 0.977 1.0 ,0.01 67.0 33910 Transporter 166 284 10 ,0.01 16.9 0.057 0.6 ,0.01 28.8 18210 GH1 b-glucosidase 164 207 10 ,0.01 17.2 0.342 0.8 ,0.01 21.7 77281 Hypothetical 158 339 5 ,0.01 34.1 0.043 0.5 ,0.01 73.5 44970 Hypothetical 157 141 15 ,0.01 10.6 0.709 1.1 ,0.01 9.5 84111 GH5-5 Endoglucanase 25.88 12.32 3.50 27.46 1.40 1.96 149 146 9 ,0.01 15.8 0.971 1.0 ,0.01 15.4 27734 Hypothetical 142 200 10 ,0.01 13.7 0.045 0.7 ,0.01 19.3 Nine highly expressed genes (.100 RPKM) exhibiting significant transcript accumulation (p,0.01;.4-fold) in ELP medium relative to Glc medium: 424549 Hypothetical 65 115 3 0.014 24.7 0.060 0.6 ,0.01 44.1 37108 Hypothetical 78 135 5 0.015 16.6 0.258 0.6 ,0.01 28.5 124522 Hypothetical 88 164 12 0.014 7.2 0.113 0.5 ,0.01 13.6 85295 Heroxidase DyP 39 145 11 0.050 3.4 0.015 0.3 ,0.01 12.8 100874 Hypothetical 295 415 63 0.018 4.7 0.072 0.7 ,0.01 6.6 125316 Hypothetical 140 201 35 0.009 3.9 0.060 0.7 ,0.01 5.7 128752 Histidine kinase 135 225 44 0.031 3.1 0.060 0.6 ,0.01 5.1 63338 PIPkin III 71 137 29 0.036 2.4 0.016 0.5 ,0.01 4.7 22173 Hypothetical 68 111 27 0.024 2.5 0.021 0.6 ,0.01 4.0

Abbreviations: SDH, short chain dehydrogenase; LPMO, lytic polysaccharide monooxygenase; AAD, aryl alcohol dehydrogenase; CDH, cellobiose dehydrogenase; OR, oxidoreductase; DyP, dye decolorizing peroxidase; PIPkin-III, heipi gigantea Phlebiopsis phosphatidylinositol-3-phosphate 5-kinase. 1Oxidoreductase is 53% identical to S. pombe AAD (Q9P7U2). 2GH61 LPMO model Phlgi1_227588 is 39-truncated. *Protein models cross listed with Tables 2 and 3. doi:10.1371/journal.pgen.1004759.t001 ooiaino Conifers of Colonization Phlebiopsis gigantea Colonization of Conifers

of the same proteins in the metabolism of pine wood extractives (Table 3, Dataset S2). Specifically, lipases Phlgi1_19028 and Phlgi1_36659 and aldehyde dehydrogenase Phlg1_115040 were more abundant in loblolly pine wood and in Avicel media relative to the same media without extractives. The role of peroxiredoxin (Phlgi1_95619) and glutathione S-transferase (Phlgi1_113065) are less clear, but transformations involving H2O2 reduction and glutathione conjugation are possible. A single MCO (Phlgi1_129839) and its corresponding transcripts, were observed to be upregulated in ELP relative to NELP. Although lacking the L2 signature common to laccases [57], the MCO4 protein may have iron oxidase activity provided that an imperfectly aligned Glu residue serves in catalysis (Text S1; Figures S20 and S21; Table S6).

Discussion The distinctive repertoire and regulation of P. gigantea genes underlie a unique and efficient system for degrading all components of conifer sapwood. Transcriptome and proteome analyses demonstrate an active system of hydrolases and LPMOs involved in the complete deconstruction of cellulose and hemicellulose. The overall enzymatic strategy is therefore similar to most cellulolytic microbes, but unlike closely related brown-rot decay Agaricomycetes such as P. placenta. With regard to ligninolysis, key genes were identified including LiPs, MnPs, CROs and GMC oxidoreductases. As in P. chrysosporium, the presence of both LiP- and GLX-encoding genes is consistent with a close physiological connection involving peroxide generation [41]. We also annotated non-class II peroxidases HTPs and DyPs some of which have been implicated in metabolism of lignin derivatives [58,59]. The distribution and expression of DyP-encoding genes are notable; with no genes present in P. chrysosporium and C. subvermispora but several highly expressed genes in T. versicolor, D. squalens [21] and P. gigantea (Table 2). Physiological roles of DyP are likely diverse, but oxidation of lignin-related aromatic compounds has been demonstrated [59]. In addition to lignin, oxidative mechanisms likely play a central role in P. gigantea cellulose attack. Of 15 LPMO-encoding genes, transcripts of six genes were regulated (.2-fold; p,0.01) and peptides corresponding to three were unambiguously identified in Figure 7. P. gigantea transcriptome. Scatterplot (A) shows the NELP- or ELP-containing media. Our inability to detect any distribution of RNA-seq RPKM values (log2) for 11,376 P. gigantea genes LPMO proteins in Avicel media (Dataset S2) suggests induction by when grown on basal salts containing acetone-extracted loblolly pine wood (ELP) or non-extracted loblolly pine wood (NELP). Lines define 2- substrates other than cellulose [60]. Beyond this, the CDH gene fold borders and best fit regression. Darkened points represent 44 was highly expressed (transcripts and protein) in LP media. The transcripts accumulating.4-fold at p,0.01. Venn diagram (B) illustrates observed coordinate expression of CDH and LPMO may reflect genes with RPKM signals.10 and upregulated.4-fold in NELP relative oxidative ‘boosting’ as recently demonstrated [19,20,47,61]. to ELP. Twenty-two genes showed significant transcript accumulation in However, we did not detect elevated transcripts or peptides NELP relative to ELP suggesting potential response to resin and pitch content. Under these stringent thresholds (p,0.01;.4-fold), only one corresponding to the two P. gigantea aldose 1-epimerase genes gene, a MCO model Phlgi1_129839, showed significant transcript even though these were previously observed in culture filtrates of accumulation in ELP relative to NELP. Additional detail appears in various white-rot fungi [21,62], including Bjerkandera adusta, Tables 1-3. Detailed methods and complete data are presented in Text Ganoderma sp, and Phlebia brevispora [17]. Thus, it seems S1 and Dataset S2. unlikely that enzymatic conversion of oligosaccharides to their b- doi:10.1371/journal.pgen.1004759.g007 anomers is necessary for efficient CDH catalysis. Softwood hemicellulose composition typically includes 15-20% galactoglucomannan while hardwoods contain 15–30% glucuronoxylan extract-induced genes, lipases Phlgi1_19028 and Phlgi1_36659 [40]. Consistent with an adaption to conifer hemicellulose, GH5_7 b- likely hydrolyze the significant levels of triglycerides. The substrate mannanases were highly expressed in both NELP and ELP cultures, specificity of aldehyde dehydrogenases such as Phlgi1_115040 is together with a GH27 a-galactosidase (Table 1). GH11 endoxylanase difficult to assess based on sequence, although several have been and CE carbohydrate esterase peptides were also detected in the pine implicated in the degradation of pine wood resins by bacteria [56]. wood-containing media, but not in Avicel cultures (Dataset S2). In Secretome patterns in media containing microcrystalline cellulose aggregate, these results demonstrate P. gigantea adaptation to conifer (Avicel) as sole carbon source generally supported the importance hemicellulose degradation.

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Figure 8. Glycoside hydrolase encoding genes show similar patterns of expression in media containing freshly ground and non- extracted loblolly pine wood (NELP) relative to the same substrate but extracted with acetone (ELP) to remove pitch and resins. Proteins (upper panel) and transcripts (lower panel) were identified by LC-MS/MS and RNA-seq, respectively. Protein identification was limited to those with.2 unique peptides after five days incubation. Transcript upregulation was limited to significant accumulation (p,0.05;.2-fold) on NELP or ELP relative to glucose-containing medium. Secretome and transcriptome experimental details and complete data are presented in Text S1 and Dataset S2. doi:10.1371/journal.pgen.1004759.g008

P. gigantea’s gene expression patterns reveal multiple strategies (Tables 1), are induced by aromatic compounds in P. chrysospor- for overcoming the challenging composition of resinous sapwood. ium [64,65]. Tolerance to monoterpenes may be mediated in part by a putative Predicted to degrade triglycerides, a total of nine lipase- ABC efflux transporter (Phlbi1_130987, Figure 9). Of the 51 ABC encoding genes were identified in the P. gigantea genome and proteins of P. gigantea, this protein is most closely related to the four of these were upregulated.2-fold (p,0.01) in LP media GcABC-G1 gene of the ascomycete Grosmannia clavigera,a compared to Glc medium (Dataset S2). Two lipases displayed pathogen of Pinus contorta [63]. The GcABC-G1 gene is similar patterns of transcript and protein upregulation on NELP upregulated in response to various terpenes and appears to be a relative to ELP (Table 3), and the pine wood extractive also key element against the host defenses. Consistent with a similar induced accumulation of these lipases in Avicel media (Table 3). function, our analysis showed the P. gigantea homolog to be Further metabolism of triglycerides is uncertain, although a upregulated.4.9-fold (p = 0.02) in NELP relative to ELP media putative glycerol uptake facilitator (Phlbi1_99331) was highly (Dataset S2). Other transcripts accumulating in NELP-derived expressed (RPKM value of 2532) and significantly (p,0.02) mycelia included three CYPs (Table 2) potentially involved in the upregulated (2.1-fold) in NELP compared to ELP (Dataset S2). hydroxylation of diterpenoids and related resin acids [55]. Fatty acids activation and b-oxidation can be inferred by the Differential regulation also implicates glutathione S-transferase, expression of fatty acid CoA ligase (Phlgi1_107548, aldehyde dehydrogenase and peroxiredoxin in the transformation Phlgi1_126556, Phlgi1_89325), b-ketothiolase (Phlgi1_27649, and detoxification of extractives (Table 2). Three putative Phlgi1_130767), and fatty acid desaturase (Phlgi1_100083, transcription regulators were similarly regulated (Table 3). Alde- Phlgi1_115799). Upregulation of a mitochondrial malate dehy- hyde dehydrogenase- and AAD-encoding genes, some of which drogenase (Phlgi1_22176, Table 3), together with relatively high are upregulated in P. gigantea LP cultures relative to Glc cultures transcript levels of other TCA cycle components (citrate synthases

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Table 2. Transcripts accumulating.4-fold in non-extracted loblolly pine wood (NELP) relative to extracted loblolly pine wood (ELP).1

RPKM Ratio

PRO ID3 Putative function Comments NELP ELP NELP/ELP Probability

385265 Hypothetical 174.01 10.84 16.058 0.001 20514* Hytochrome P450 CYP512B 368.06 29.90 12.310 ,0.001 118355 Hypothetical 72 aa; IVS 132.48 10.98 12.068 ,0.001 21241 GH11 endo-b-1,4-xylanase 133.85 12.68 10.559 0.006 118300 Hypothetical 84 aa 210.69 28.40 7.419 ,0.001 119094 Cytochrome P450 CYP5148A 180.08 31.01 5.808 0.008 128107 Hypothetical 53.61 10.25 5.229 0.001 35298 Hypothetical 204.88 40.46 5.064 ,0.001 75047 Hypothetical 178.94 35.38 5.057 ,0.001 116265 Hypothetical 61.31 12.38 4.954 0.002 99997 Hypothetical 162.61 33.31 4.882 0.008 19184 Cytochrome P450 CYP5136A 99.22 20.33 4.880 0.002 19877 Hypothetical 57.37 11.83 4.849 0.007 123837* Zn finger domain protein 8222.76 1745.42 4.711 0.002 340250 HMG-Box transcription factor 295.72 63.02 4.693 ,0.001 88507* GH18 Chitinase CBM5 409.32 87.55 4.675 0.002 122504 Hypothetical 245.20 55.28 4.436 0.006 125213* GH61 LPMO2 1290.80 305.62 4.223 ,0.001 36458 HMG-Box transcription factor 256.76 61.38 4.183 0.001

1Listing limited to genes with RPKM values.10 and high confidence differential expression (p,0.01). Complete listings for 11,892 genes provided in Dataset S2. Abbreviations: aa, amino acids; CYP, cytochrome P450; IVS, long intervening sequence in gene model; CBM, carbohydrate binding module; 2Truncated gene model predicts incomplete protein (117aa). 3Nineteen of 22 accumulating in NELP relative to ELP as illustrated in Figure 4B. Three additional upregulated genes were associated with LC-MS/MS-detected proteins and listed in Table 3. Proteins with asterisks are also listed in Table 2. doi:10.1371/journal.pgen.1004759.t002

Phlgi1_126205, Phlgi1_100215; 2-oxoglutarate dehydrogenase, designated as hypothetical (Table 2; Dataset S2). Three of these Phlgi1_126652) may complete fatty acid oxidation. In this featured predicted secretion signals and peptides were detected in connection, we also observed high expression of isocitrate one case. In the absence of biochemical characterization and/or lyase (Phlgi1_21482, Phlgi1_93159) and malate synthase genetic evidence, assigning function to these genes represents a (Phlgi1_27815), which partially explain oxalate accumulation major undertaking. Nevertheless, high throughput transcript and [66] and strongly support an active glyoxylate shunt [45,67] secretome profiling substantially filtered the number of potential (Figure 9). Upregulation of glycoside hydrolases, transcription targets from a genome-wide estimate of 4744 ‘hypothetical’ genes factors, cyclophilins, ATP synthase and ribonuclease may also to the more manageable numbers reported here. More broadly, reflect broad shifts in metabolism or reduced accessibility of the the results advance understanding of the early and exclusive unextracted substrate (Tables 2 and 3). colonization of coniferous wood by P. gigantea and also provide a Beyond genetic regulation, certain constitutively expressed framework for developing effective wood protection strategies, genes are also likely involved in the degradation of all plant cell improving biocontrol agents and identifying useful enzymes wall components, including complex resins and triglycerides. For [6,9,10]. example MOX (Phlgi1_120749) is among the most abundant transcripts in both NELP and ELP (Dataset S2), suggesting an Methods important role in H2O2 production associated with lignin demethylation [53]. Extracellular peroxide generation is key to Wood colonization assays peroxidase activity, and MOX fulfills this role along with CRO, Wood wafers (1 cm by 1 cm by 2 mm) were cut from the AAO, and P2O. Along these lines, we also observed high sapwood of aspen (Populus tremuloides), pine (P. taeda) and spruce extracellular protein levels of DyP (Phlgi1_122124) under all (Picea glauca) and sterilized by autoclaving. Following inoculation culture conditions. by contact with mycelium growing on malt extract agar (15 g malt Most problematic, many P. gigantea genes and proteins extract [Difco, Detroit, MI] and 15 g agar per liter of water) in exhibited little or no homology to NCBI NR or Swiss-Prot entries. Petri dishes, colonized wafers were harvested 30, 60 and 90 days. Some of these ‘hypothetical’ or ‘uncharacterized’ proteins are Noninoculated wood wafers placed on the same media in Petri undoubtedly important, particularly those that are highly dishes served as controls. Wafers were removed 30, 60 or 90 days expressed, regulated and/or secreted. For example, of 92 genes later, weighed and percent weight loss was determined. Additional upregulated (.2-fold; p,0.01) in NELP relative to ELP, 51 were wafers were removed at the same time period, immediately frozen

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Figure 9. Glyoxalate shunt and proposed relationship to lipid oxidation when P. gigantea is cultivated on wood-containing media (ELP or NELP) relative to Glc medium. Enzymes encoded by upregulated genes are black highlighted and associated with thickened arrows. Abbreviations: ABC-G1, ABC transporter associated with monoterpene tolerance; ADH/AO, Acyl-CoA dehydrogenase/oxidase; AH, Aconitate hydratase; CoA ligase, long fatty acid-CoA ligase; DLAT, Dihydrolipoyllysine-residue acetyltransferase; DLST, Dihydrolipoyllysine-residue succinyltransferase; EH, Enoyl-CoA hydratase; FDH, Formate dehydrogenase; FH, Fumarate hydratase; KT, Ketothiolase (acetyl-CoA C-acyltransferase); HAD, 3-Hydroxyacyl-CoA dehydrogenase; ICL, Isocitrate lyase; IDH, Isocitrate dehydrogenase; MDH, Malate dehydrogenase; MS, Malate synthase; ODH, Oxoglutarate dehydrogenase; OXA, Oxaloacetase; OXDC, Oxalate decarboxylase; OXO, Oxalate oxidase; PC, Pyruvate carboxylase; PDH, Pyruvate dehydrogenase; PEP, Phosphoenolpyruvate; PEPCK, Phosphoenolpyruvate carboxykinase; PEPK, Phosphoenolpyruvate kinase; SDH, succinate dehydrogenase. See Dataset S2 for detailed gene expression data. doi:10.1371/journal.pgen.1004759.g009

Pine emPAI values RNA-seq 5 days Transcript ratio (R) & Probabilities (Prob) emPAI values

Days in NELP Days in ELP RPKM NELP/Glc NELP/ELP ELP/Glc 5 day Avicel

PRO id Putative function 5 7 9 5 7 9 NELP ELP Glc Prob R Prob R Prob R + extr - extr

19028 Lipase 6.86 1.20 0.76 1.79 1.92 1.34 1903 274 22 0.001 87.7 0.000 7.0 0.007 12.6 2.24 0.80 126044 Cyclophilin 6.77 0.70 0.34 3.63 0.58 0.54 272 56 195 0.637 1.4 0.007 4.9 0.124 0.3 5.89 8.78 22176 Malate dehydrogenase 0.44 0.00 0.07 0.14 1.34 0.27 64 15 51 0.705 1.3 0.003 4.2 0.092 0.3 3.70 7.12 26602 GH17 b-(1-6) endohydrolase 0.00 0.09 0.09 0.00 0.00 0.00 478 124 30 0.119 15.9 0.003 3.8 0.331 4.1 0.00 0.00 95619 Peroxiredoxin 0.26 0.19 0.23 0.00 0.00 0.00 98 26 108 0.773 0.9 0.001 3.8 0.023 0.2 1.35 1.35 113065 Glutathione S-transferase 0.97 0.69 0.05 0.00 0.00 0.00 52 14 45 0.569 1.2 0.000 3.6 0.023 0.3 0.00 0.00 36659 Lipase 0.93 0.00 0.12 0.00 0.00 0.00 285 86 178 0.473 1.6 0.005 3.3 0.292 0.5 0.66 0.22 33454 GH13-CBM20 a-amylase 1.23 1.44 0.67 0.78 1.34 2.16 501 186 168 0.405 3.0 0.005 2.7 0.941 1.1 0.68 0.77 115040 Aldehyde dehydrogenase 0.60 0.00 0.02 0.00 0.00 0.00 101 38 14 0.012 7.4 0.001 2.7 0.057 2.8 1.89 0.51 64365 Ribonuclease T2 0.64 0.25 0.15 0.38 1.06 0.98 80 32 18 0.015 4.5 0.009 2.5 0.107 1.8 0.69 2.31 70525 ATP synthase 0.54 0.12 0.04 0.00 0.00 0.00 22 10 29 0.246 0.8 0.004 2.2 0.020 0.3 0.91 1.37 36220 Glycoprotein 0.17 0.17 0.04 0.12 0.38 0.23 106 48 365 0.204 0.3 0.009 2.2 0.071 0.1 0.00 0.00 19596 Uncharacterized protein 0.97 0.69 0.40 0.45 0.00 0.00 1937 900 2056 0.807 0.9 0.002 2.2 0.035 0.4 0.00 0.00 129839 Multicopper oxidase (MCO4) 1.45 1.03 0.25 2.61 8.79 6.10 40 209 180 0.091 0.2 0.010 0.2 0.852 1.2 0.50 1.02

1Listing limited to genes with RPKM values.10 and, in comparisons of NELP and ELP cultures, with high confidence of differential expression (p,0.01). Transcript values for cultures grown on microcrystalline cellulose (Avicel) as sole carbon source unavailable. The composition of loblolly pine wood extract (extr) is listed in Text S1. Complete listings for 11,892 genes provided in Dataset S2. doi:10.1371/journal.pgen.1004759.t003 heipi gigantea Phlebiopsis ooiaino Conifers of Colonization Phlebiopsis gigantea Colonization of Conifers to 220uC and prepared for scanning electron microscopy as Figure S3 Ten genes encoding enzymes potentially involved in previously described [68]. lipid metabolism contributing to PC1 and PC2 values in Figure 3B. The x-axis designates each enzyme family and y-axis Sequencing and annotation indicates the squared rotation values for PC1 and PC2. The genome was sequenced using Illumina and annotated using (EPS) the JGI Annotation Pipeline [69]. Assembly and annotations are Figure S4 Phylogenetic analysis of opsin genes from P. gigantea available from JGI portal MycoCosm [38] and deposited to (Phlgi), C. subvermispora (Cersu), P. placenta (Pospl), A. nidulans DDBJ/EMBL/GenBank under accession AZAG00000000. The (AN), Sordaria macrospora (SM) and Neurospora crassa (NCU). version described in this paper is version AZAG01000000. The The evolutionary history was inferred using the Minimum completeness of the P. gigantea genome was assessed by finding Evolution method [78]. The bootstrap consensus tree inferred 99.1% of CEGMA proteins conserved across sequenced genomes from 500 replicates (MEGA4) is taken to represent the of eukaryotes [70](Text S1; Tables S1, S2). evolutionary history of the taxa analyzed (MEGA4). Branches corresponding to partitions reproduced in less than 50% bootstrap RNA-seq replicates are collapsed. The percentage of replicate trees in which Mycelium was derived from triplicate cultures of 250 ml basal the associated taxa clustered together in the bootstrap test (500 salts containing: i. 1.25 g freshly-harvested, ground (1mm mesh) replicates) are shown next to the branches (MEGA4). The tree is loblolly pine wood that had been ‘spiked’ with acetone and drawn to scale, with branch lengths in the same units as those of thoroughly dried (NELP); or ii. the same material following the evolutionary distances used to infer the phylogenetic tree. The extended acetone extraction in a Soxhlet apparatus and drying evolutionary distances were computed using the Poisson correction (ELP). The composition of the extract (Text S1) was determined method [79] and are in the units of the number of amino acid by GC-MS [51]. Duplicate cultures of basal salts medium with substitutions per site. The ME tree was searched using the Close- glucoseassolecarbonsourceservedasareference.After5days Neighbor-Interchange (CNI) algorithm [4] at a search level of 1. incubation, total RNA was purified from frozen mycelium as The Neighbor-joining algorithm [80] was used to generate the described [22,71]. Multiplexed libraries were constructed and initial tree. All positions containing gaps and missing data were sequenced on an Illumina HiSeq2000. DNAStar Inc (Madison, eliminated from the dataset (Complete deletion option). There WI) modules SeqNGen and Qseq were used for mapping reads were a total of 183 positions in the final dataset. Phylogenetic and statistical analysis. Transcriptome data was deposited to the analyses were conducted in MEGA4 [81]. NCBI Gene Expression Omnibus (GEO) database and assigned (EPS) accession GSE53112 (Reviewer access via http://www.ncbi. Figure S5 Phylogenetic analysis of putative photoreceptors of nlm.nih.gov/geo/query/acc.cgi?token=ilovmswixtajjez&acc=GSE Ceriporiopsis subvermispora (Cersu), Phlebiopsis gigantea (Phlgi), 53112). Experimental details are provided in Text S1 and all Postia placenta (Pospl), Cryptococcus neoformans (CN), Laccaria transcriptome analyses are summarized in Dataset S2. bicolor (LB), Phycomyces blakesleeanus (PB), Neurospora crassa (NCU), Aspergillus nidulans (ANIDU) and Trichoderma reesei Secretome analysis (TR). Along with the species, the name is given of the respective With minor modification, NanoLC-MS/MS analysis identified protein (if known) and the GenBank accession number or protein extracellular proteins in culture filtrates as described [22,72]. For ID in JGI genome databases. each of the two woody substrates (e.g NELP and ELP), cultures (EPS) were harvested after 5, 7 and 9 days. Mass spectrometric protein Figure S6 Alignment of the homologous region comprising the identifications were accepted if they could be established at PAS/LOV domain (NCRFLQ) in photoreceptor orthologues. greater than 95.0% probability within 0.9% False Discovery Rate LOV signatures are highlighted. and contained at least two identified peptides. Protein probabil- (EPS) ities were assigned by the Protein Prophet algorithm [73]. To verify the effects of pine wood extractives in a well-defined Figure S7 Genomic locus comprising the cluster of response substrate, media containing microcrystalline cellulose (Avicel) regulator genes. were also employed [22,45,74]. Filtrates from these cultures, with (EPS) or without addition of loblolly pine wood acetone extract, were Figure S8 Homology models for the molecular structures of collected after 5 days and analyzed. Approximate protein class II heme peroxidases from the P. gigantea genome. abundance in each of the cultures was expressed as the number Ligninolytic peroxidases, including LiP models - A) 150531 of unique peptide and the exponentially modified protein peroxidase, B) 121662 peroxidase and C) 30372 peroxidase - abundance index (emPAI) value [52] (See Text S1 for detailed harboring an exposed tryptophan potentially involved in oxidation methods). of high redox-potential substrates, and MnP models - D) 75566 peroxidase, E) 75572 peroxidase, F) 115591 peroxidase, G) Supporting Information 115592 peroxidase and H) 117668 peroxidase - harboring a 2+ Figure S1 Vista dot plot illustrating syntenic relationship putative Mn oxidation site (formed by two glutamates and one aspartate); and I) manually curated GP (32509). Note that an between 12 longest scaffolds of P. gigantea and P. chrysosporium. alanine and an asparagine residues in the LiP models occupy the (EPS) position of the catalytic glutamate and aspartate involved in Mn2+ Figure S2 Top 20 families of contributing to PC1 and PC2 oxidation by MnP, and a serine residue in the MnP models values in Figure 3A. The x-axis designates each enzyme family occupies the position of the putative catalytic tryptophan and y-axis indicates the squared rotation values for PC1 and PC2. characterizing LiP. The amino acid numbering refers to putative As shown in Figure 2, the PC2 value mainly separated the white- mature sequences, after manual processing of their peptide and brown-rot fungi. sequences. (EPS) (EPS)

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Figure S9 Dendrogram showing evolutionary relationships functional classification. A) Short, long and extralong MnPs have a among 478 basidiomycete heme peroxidases, including structur- Mn2+-oxidation site formed by two glutamic and one aspartic al-functional classification based on Ruiz-Duen˜as et al. (2) residues, and differ in the length of the C-terminal tail; B) LiPs (GeneBank and JGI references in parentheses and P. gigantea contain a catalytic tryptophan, with the only exception of TRACE- genome references on yellow background). Amino-acid sequence LiP being the ‘‘unique’’ ligninolytic peroxidase with a catalytic comparisons as Poisson distances and clustering based on tyrosine (3); C) VPs harbor the catalytic sites described above for UPGMA and "pairwise deletion" option of MEGA5 [81]. both MnPs and LiPs; D) GPs do not contain any of the above two Compressed sub-trees are shown to facilitate the P. gigantea catalytic sites; and E) atypical MnPs and VPs lack one of the three peroxidases analysis. Numbers on branches represent bootstrap acidic residues forming the Mn2+-oxidation site. The analysis is values (based on 1000 replications) supporting that branch; only described in Figure S9. Fungal abbreviations: AGABI, the values $50% are presented. Fungal abbreviations: Agaricus bisporus; AURDE, Auricularia delicata; BJEAD, Bjer- AGABI, Agaricus bisporus; AURDE, Auricularia delicata; kandera adusta; BJESP, Bjerkandera sp; CERRI, Ceriporiopsis BJEAD, Bjerkandera adusta; CERSU, Ceriporiopsis subvermis- rivulosa; CERSU, Ceriporiopsis subvermispora-B; CERUN, Cer- pora-B; COPCI, Coprinopsis cinerea; DACSP, Dacryopinax sp.; rena unicolor; COPCI, Coprinopsis cinerea; COPDI, Coprinellus DICSQ, Dichomitus squalens v1.0; FOMME, Fomitiporia medi- disseminatus; DICSQ, Dichomitus squalens v1.0; FOMME, terranea v1.0; FOMPI, Fomitopsis pinicola SS1 v1.0; IZU, Fomitiporia mediterranea v1.0; FOMPI, Fomitopsis pinicola SS1 basidiomycete IZU-154; LACBI, Laccaria bicolor v2.0; LENED, v1.0; GANAP, Ganoderma applanatum; GANAU, Ganoderma Lentinula edodes; PHACH, Phanerochaete chrysosporium; australe; GANFO, Ganoderma formosanum; GANLU, Ganoderma PHASO, Phanerochaete sordida; PHLBR, Phlebia brevispora lucidum; GANSP, Ganoderma sp.; HETAN, Heterobasidion HHB-7030 SS6 v1.0; PHLGI, Phlebiopsis gigantea; POSPL, annosum v2.0; IZU, basidiomycete IZU-154; LACBI, Laccaria Postia placenta; PUNST, Punctularia strigosozonata v1.0; bicolor v2.0; LENED, Lentinula edodes; PHACH, Phanerochaete SERLA, Serpula lacrymans; SPOSP, Spongipellis sp.; STEHI, chrysosporium; PHASO, Phanerochaete sordida; PHLBR, Phlebia Stereum hirsutum FP-91666 SS1 v1.0; TRACE, Trametopsis brevispora HHB-7030 SS6 v1.0; PHLGI, Phlebiopsis gigantea; cervina; TREME, Tremella mesenterica; USTMA, Ustilago PHLRA, Phlebia radiata; PLEER, Pleurotus eryngii; PLEOS, maydis. Most of the sequences included in the dendrogram were Pleurotus ostreatus; PLEPU, Pleurotus pulmonarius; PLESA, obtained from the analysis of fungal genome sequences. The Pleurotus sapidus; POSPL, Postia placenta; PUNST, Punctularia genome version from which the peroxidase sequence was obtained strigosozonata v1.0; SPOSP, Spongipellis sp.; STEHI, Stereum is in some cases indicated as v1.0 and v2.0. Peroxidase hirsutum FP-91666 SS1 v1.0; TAICA, Taiwanofungus camphor- abbreviations: i) GP, generic peroxidase; ii) MnP-short, MnP- atus; TRACE, Trametopsis cervina; TRAVE, Trametes versicolor; long and MnP-extralong, three different mangenese peroxidase WOLCO, Wolfiporia cocos MD-104 SS10 v1.0. GeneBank and (MnP) subfamilies including a typical Mn(II)-oxidation site, formed JGI references are shown in parentheses and P. gigantea genome by two glutamates and one aspartate residues, and differing in the references on yellow background. length of their C-terminal tails; iii) LiP, lignin peroxidase (PDF) harboring an exposed tryptophan residue located at the same Figure S13 Dendrogram focused on DyP peroxidases (a total of position described for the catalytic Trp171 of P. chrysosporium 64) showing evolutionary relationships. The analysis is described in LiP; iv) VP, versatile peroxidase including a Mn(II)-oxidation site Figure S7. Fungal abbreviations:AURAU,Auricularia auricula- like in MnP, and a catalytic tryptophan like in LiP; v) VP-LiP judae;AURDE,Auricularia delicata;BJEAD,Bjerkandera adusta; intermediate states, two Ceriporiopsis subvermispora peroxidases COPCI, Coprinopsis cinerea; DICSQ, Dichomitus squalens v1.0; occupying an intermediate position between typical LiPs and VPs FOMME, Fomitiporia mediterranea v1.0; GANLU, Ganoderma according to their structural and catalytic properties [22]; and vi) lucidum;GANSP,Ganoderma sp.; HETAN, Heterobasidion annosum MnP-atypical and VP-atypical, MnP and VP lacking one of the v2.0; LACBI, Laccaria bicolor v2.0;MARSC,Marasmius scorodo- three acid residues forming the typical Mn(II)-oxidation site nius;MELLA,Melampsora laricis-populina v1.0;PHLBR,Phlebia present in MnP and VP (Glu35/36, Glu39/40 and Asp179/175 in brevispora HHB-7030 SS6 v1.0; PHLGI, Phlebiopsis gigantea; P. chrysosporiun MnP/P.eryngii VP). PLEOS, Pleurotus ostreatus;POSPL,Postia placenta;PUNST, (PDF) Punctularia strigosozonata v1.0; STEHI, Stereum hirsutum FP-91666 Figure S10 Homology models for the molecular structures of SS1 v1.0; TERAL, Termitomyces albuminosus;TRAVE,Trametes heme-thiolate peroxidases (HTPs) from the P. gigantea genome. versicolor. GenBank and JGI references are shown in parentheses and A) Phlgi1_131735 peroxidase, B) Phlgi1_18201 peroxidase, C) P. gigantea genome references on yellow background. Phlgi1_19534 peroxidase and D) Phlgi1_104428 peroxidase, (PDF) including proximal cysteine residue acting as the fifth heme iron Figure S14 Multiple alignments of aryl alcohol oxidases (AAO) ligand and a few more amino acid residues of the active center. sequences from P. gigantea (Phlgi128071, Phlgi121514) and P. (EPS) eryngii (AAOpe) [82]. Highly conserved histidine active site Figure S11 Homology models for the molecular structures of residues [83] are in red. Bottom lines show conserved motifs in DyP peroxidases from the P. gigantea genome. A) Phlgi1_122124 GMC family [84]. Obtained using Clustal W [85].The peroxidase (old Phlgi1_78526 peroxidase), B) Phlgi1_71660 Phlgi128071 and Phlgi121514 models conserve the substrate- peroxidase, C) Phlgi1_85295 peroxidase and D) Phlgi1_125681 binding pocket reported for AAO from P. eryngii and the motifs in peroxidase, including amino acid residues located at the proximal GMC family. and distal sides of the heme active site involved in catalysis. (EPS) (EPS) Figure S15 Multiple alignments of methanol oxidase (MOX) Figure S12 Dendrogram focused on class II heme peroxidases (a sequences from P. gigantea (Phlgi120749, Phlgi108516 total of 219) showing evolutionary relationships and structural- and Phlgi72751) and G. trabeum (ABI14440.1)[53]. Highly conserved

PLOS Genetics | www.plosgenetics.org 16 December 2014 | Volume 10 | Issue 12 | e1004759 Phlebiopsis gigantea Colonization of Conifers histidine/asparagine active site residues [84] are in red. Bottom lines Figure S22 Phylogenetic analysis and subfamily assignments of show conserved motifs in GMC family [84]. Obtained using Clustal W GH5 protein models of P. gigantea (Phlgi), H. annosum (Hetan) [85]. The MOX models conserve motifs in GMC family. and Stereum hirsutum (stehi). (EPS) (EPS) Figure S16 Multiple alignments of cellobiose dehydrogenase (CDH) Figure S23 Phylogenetic analysis of LPMO proteins of P. sequence from P. gigantea (model Phlgi99876) and Gelatoporia gigantea, C. subvermispora, and P. Chrysosporium. subvermispora (ACF60617). Highly conserved active site residues are in (EPS) red [86]. Obtained using Clustal W [85]. Figure S24 Phylogeny and differential expression of carbohy- (EPS) drate esterase family 1 (CE1) genes. CDS sequences were obtained Figure S17 Multiple alignments of pyranose oxidse (POX) from each genome database according to assigned protein IDs. sequences from P. gigantea (Phlgi130349), Peniphora sp. Incomplete CDS sequences (partial fragments) were eliminated (AAO13382.1) and Trametes ochracea (AAP40332.1). Obtained using from the analysis. For each CE family, a multiple alignment was Clustal W [85]. performed using MegAlign version 10 software. The phylogenetic (EPS) tree was then constructed from the multiple alignment using Clustal W [85]. Numbers at the nodes represent bootstrap values, Figure S18 Multiple alignments of glucose oxidase (GOX) based on 1000 replications. Species: Aurde, Auricularia delicata sequences from P. gigantea (Phlgi128108), B. fuckeliana SS-5; Conpu, Coniophora puteana; CC1G, Coprinopsis cinerea; (CDA88590.1) and C. immitis (EAS27606) and A. niger Dicsq, Dichomitus squalens; Fomme, Fomitiporia mediterranea; (AAF59929.2) [1]. Highly conserved histidine active site residues Fompi, Fomitopsis pinicola FP-58527 SS1; Glotr, Gloeophyllum [84] are in red. Bottom lines show conserved motifs in GMC trabeum; Hetan, Heterobasidion annosum; Lacbi, Laccaria bicolor; family [84]. Obtained using Clustal W [85]. Phaca, Phanerochaete carnosa HHB-10118; Phach, Phanerochaete (EPS) chrysosporium RP78; Phlgi, Phlebiopsis gigantea; Punst, Punctu- Figure S19 Multiple alignments of eight putative aryl alcohol laria strigosozonata; Schco, Schizophyllum commune; Serla, dehydrogenase (AAD) sequences from P. gigantea (Phlgi1) and P. Serpula lacrymans S7.3; Stehi, Stereum hirsutum FP-91666 SS1; chrysosporium (AAA61931.1)[36]. Obtained using Clustal W [85]. Trave, Trametes versicolor; Wolco, Wolfiporia cocos MD-104 (EPS) SS10. Ecology: W, white rot; B, brown rot; M, mycorrhiza; S, non-wood decay saprotroph. Location of comprised CBM1 was Figure S20 Phylogenetic tree of multicopper oxidases from indicated as N or C-terminal. Differential regulation of Phlgi CE Acremonium sp. (Acr), Aspergillus nidulans (Ani), Cryptococcus transcripts between the cultivations tested in this study were also neoformans (Cne), Coprinopsis cinerea (Cci), Pleurotus ostreatus (Pos), indicated as up- (U) or down- (D) regulated. Asterisk was Phanerochaete carnosa (Pca), Phanerochaete chrysosporium (Pch), accompanied if the p value was ,0.05. Possible CE1 gene Phanerochaete flavido-alba (Pfa), Phlebiopsis gigantea (Pgi), Postia Phlgi_121418 was excluded as the model was severely truncated placenta (Ppl), Saccharomyces cerevisiae (Sce), Schizophyllum commune (91 amino acid residues). (Sco), Serpula lacrymans (Sla), Sporobolomyces roseus (Sro), Tremella (PDF) mesenterica (Tme), and Ustilago maydis (Uma). Alignments were produced in program ClustalW, manually adjusted in Genedoc and Figure S25 Phylogeny and differential expression of CE4 genes. computed in MEGA4.0 for phylogenetic tree production (neighbour- Analysis and abbreviations as in Figure S24. joining, bootstrap values 500). ID numbers refer to protein models in (PDF) the JGI MycoCosm (http://genome.jgi-psf.org/programs/fungi/ Figure S26 Phylogeny and differential expression of CE8 genes. index.jsf), other accession numbers to the NCBI database (http:// Analysis and abbreviations as in Figure S24. Possible CE8 gene www.ncbi.nlm.nih.gov/), specific names to proteins specified with Phlgi_132681 was excluded as the model was severely truncated accession numbers in Hoegger et al. [29] and Lettera et al. [87]. Blue (79 residues). coloring marks enzymes with laccase activity experimentally shown, (PDF) lightbrownenzymeswithprovenferroxidase activity, purple enzymes with ascorbate activity, olive enzymes acting in fungal pigment Figure S27 Phylogeny and differential expression of CE9 genes. synthesis, and two colors dual enzymatic activities with the left color Analysis and abbreviations as in Figure S24. marking the respective major performance ([88,89] and references in (PDF) the review of Ku¨esandRu¨hl 2011 [90]). Proteins from P. gigantea are Figure S28 Phylogeny and differential expression of CE12 highlighted in red. genes. Analysis and abbreviations as in Figure S24. (EPS) (PDF) Figure S21 Sequence alignment for four regions of S. cerevisiae Figure S29 Phylogeny and differential expression of CE15 ferroxidase Fet3 with corresponding regions of enzymes of P. genes. Analysis and abbreviations as in Figure S24. gigantea and Phanerochaete species. Marked in yellow are residues (PDF) that in Fet3 of S. cerevisiae are critical for Fe2+ binding and the electron-transfer pathway (for references see [90]). Three groups of Figure S30 Phylogeny and differential expression of CE16 enzymes become obvious: i) the Fet3-type ferroxidases; ii) within the genes. Analysis and abbreviations as in Figure S24. Possible CE16 gene Phlgi_73119 was excluded as the model was severely cluster of ferroxidases/laccases one subgroup that is more similar to truncated (95 residues). the Fet3-type ferroxidases to which the ferroxidase Mco1 of P. (PDF) chrysosporium belongs to; and iii) one more distinct subgroup that misses three amino acids in the second region of importance for Figure S31 Phylogenetic tree of the cytochrome P450 proteins binding pocket formation and to which P. favido-alba bona fide (P450ome) in P. gigantea. Tree was constructed using 124 P450 laccase PfaL belongs. sequences (which were full-length or near full-length) and (EPS) evolutionary history was inferred using bootstrap Neighbor-

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Joining method. Phylogenetic analyses were conducted using Table S5 Expression of P. gigantea GMC oxidoreductases in MEGA4 [81]. The P450 listing in the tree is based on the solvent extracted lodgepole pine wood (ELP) and non-extracted corresponding protein ID with the CYP name in parenthesis. lodgepole pine wood (NELP). P450s that belong to a new subfamily are indicated with the (DOCX) abbreviation NS. Table S6 Multicopper oxidases of P. gigantea. (EPS) (DOCX) Figure S32 Comparative evolutionary analysis of the P450omes Table S7 Glycoside hydrolase comparisons of brown-rot (BR) of P. gigantea and Phanerochaete species (P. chrysosporium and P. and white-rot (WR) fungi. carnosa). Clan level comparison was made for this analysis. (DOCX) (EPS) Table S8 Carbohydrate esterase comparisons of brown-rot (BR) Figure S33 Multiple alignment of representative protein sequences and white-rot (WR) fungi. of P. gigantea hydrophobins. Sequences were aligned using MUSCLE (DOCX) alignment tool implemented in Molecular Evolutionary Genetic Analysis software (MEGA 5.0). MUSCLE was chosen because it is Table S9 Polysaccharide lyase comparisons of brown-rot (BR) computationally more suitable for multiple sequence alignments and and white-rot (WR) fungi. gives a better accuracy than the conventional CLUSTAL alignment (DOCX) [91]. The aligned protein sequences were viewed with the Biological Table S10 Protein features and regulation of lytic polysaccha- sequence alignment editor (Bioedit), windows 95/98/NT/2K/XP. ride monooxygenases (LPMO) in P. gigantea. Identical amino acid residues are marked in black, conserved cysteine (DOCX) residues are marked with asterisks. (EPS) Table S11 Properties of the transcription factors for which binding sites have been detected in CAZymes that are significantly Figure S34 The evolutionary history of P. gigantea hydro- regulated during growth on ground pine wood. phobins with a selected set of closely related basidiomycetes and (DOCX) Acremonium alcalophilum, an ascomycete. Evolutionary related- ness was inferred using the Neighbor-Joining method [80]. The Table S12 Transcript levels of potential regulators in P. percentage of replicate trees in which the associated taxa clustered gigantea cultures. together in the bootstrap test (500 replicates) are shown next to the (DOCX) branches. Branches with blue colour represent hydrophobin Table S13 Overview of the P. gigantea P450ome and sequences from P. gigantea and branches without support values comparison with P. chrysosporium and P. carnosa. are less than 50%. The tree is drawn to scale, with branch lengths (DOCX) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were Table S14 Clan-, family-, and subfamily- level classification of computed using the Poisson correction method [79], and are in the P450ome of P. gigantea and comparison with P. chrysospor- the units of the number of amino acid substitutions per site. The ium and P. carnosa. analysis involved 174 amino acid sequences. All ambiguous (DOCX) positions were removed for each sequence pair. There were a Table S15 Expression profile of P. gigantea P450ome. total of 155 positions in the final dataset. Evolutionary analyses (DOCX) were conducted in MEGA5 [92]. Fungal species IDs: |Cersu| (Ceriporiopsis subvermispora), |Phlgi| (Phlebiopsis gigantea), Table S16 Products of annotated putative secondary metabolite |Phchr|(Phanerochaete chrysosporium), |Gansp| (Ganoderma genes in the P. gigantea genome. sp.), |Phlbr| (Phlebia brevispora), |Serla_varsha|(Serpula lacry- (DOCX) mans), |Wolco| (Wolfiporia cocos), |Hetan| (Heterobasidion Table S17 P. gigantea hydrophobin models. annosum), |Schco| (Schizophyllum commune), |Copci| (Copri- (DOCX) nopsis cinerea), |Lacbi| (Laccaria bicolor), |Ustma| (Ustilago maydis), |Acral| (Acremonium alcalophilum). The tree is rooted at Table S18 Fungal species and hydrophobin gene number used Acremonium alcalophilum, representing class II of hydrophobins. for phylogenomics analysis. (EPS) (DOCX) Figure S35 Chromatogram of loblolly pine wood acetone Table S19 RNAseq analysis of P. gigantea hydrophobins. extract. (DOCX) (EPS) Table S20 P. gigantea ABC models. Table S1 Genome assembly. (DOCX) (DOCX) Table S21 Chemical composition of lipids from Loblolly pine Table S2 Annotation. wood (Pinus taeda). (DOCX) (DOCX) Table S3 Transcript profiles of response regulator genes Text S1 Detailed description of methods and annotated gene clustered on scaffold. families. (DOCX) (DOCX) Table S4 Copper radical oxidases (CROs) of P. Gigantea. Dataset S1 Number and distribution of genes used for PCA. (DOCX) (XLSX)

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Dataset S2 Complete listing of P. gigantea protein models and the data: CH TI KI MSa HSuz EM PF FJRD BHel PC LFL MSc ISD expression data. CPK JAG PK FSJ JG KS JY ACM AK FOA GL DH HSun JR AG EL (XLSX) KB RR IVG BHen UK RMB ATM SFC RAB DC. Wrote the paper: CH KI MSa HSuz EM PF FJRD PC LFL MSc ISD CPK PK FSJ KS JY AK FOA GL DH RR IVG BHen UK RMB ATM SFC RAB DC. Author Contributions Conceived and designed the experiments: ATM SFC RAB DC. Performed the experiments: CH JAG GS SSB EL JR AG SFC RAB DC. Analyzed

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